Friday, August 22, 2008

Sequencing and Identification of Protein

  1. Sample Preparation

    Hundreds and thousands of copies of single cell type is made from which protein can be extracted.

    Get Cell: Cell of single type is grown, obtained from biopsy or body fluids

    Culture Cell: Cells are then placed in growth medium in petridish. Cells feed and multiply. Now base of the petridish is covered with thousands of cells.

    Make More Copies: Cells are again divided in more petridishes to get more copies of cells. Millions of copies are produced. Then the cells are scraped from petridish base and put it in test tube

    Add Detergent: Detergent added ruptures outer membrane of cell membrane. Now the test tube contains proteins along with cell debris.

    Spin: The solution is know centrifuged to separate proteins form cell debris like cell membrane, cytoskeleton. After centrifugation the test tube contains cell debris at bottom and protein above.

  2. Separation

    A single cell has millions of protein type. Hence there separation is necessary.

    2D Electrophoresis: Separation takes place along line i.e. in one dimension and further separated over an area i.e. in second dimension.

    First Dimension Separation: Protein is placed in gel strip. The gel strip has pH gradient that ranges from acidic to alkali. The proteins are color coded based on pI levels i.e. isoelectric point.

    Then voltage is applied which moves the protein into location where electric charge of protein balances with gradient. Proteins are now organized according to pI along line i.e. in one dimension.

    Second Dimension Separation: A solution is added to proteins to give it negative charge. Then it is transferred to gel sheet in a tray. Voltage is applied. Proteins get separated according to size.

    Transfer: Proteins are now organized by pI in one dimension and size in second dimension. But still proteins are not completely separated. A single dot may contain 2 to more different protein type. Now the dots are cut out and transferred to 96 well plate or test tubes. This is done either in hand or using computer through robotic arm.

  3. Ionization

    Cut the protein into smaller pieces (peptides), and then provide a steady stream of peptides to the mass spectrometer.

    Cleavage of Proteins: Done by enzyme protease. In avg. peptides of 20 amino acids long chain are produced. Then the mixture is evaporated leaving dry peptides. Then another liquid is added that dissolves these dry peptides.

    Separation of Peptides: Then the peptides with solution are carried into liquid chromatography (LC). LC has tiny spheres which attracts the peptides. Then the peptides are freed from spheres by changing the content of solution slowly.

    Spraying of Peptide ions: Peptides then reach end of the cone shaped tube due to electric field produced. Peptides pulled out of the tube into air by electric field. The solvents evaporate from the airborne droplets. Positive charge is left behind on peptide. Then positively charged peptide moves towards negatively charged mass spectrometer.

  4. Mass Spectroscopy

    It measures the mass of the peptides and peptide fragments. From it sequence of amino acid can be identified. It consists of two quadrupole analyzer and time of flight(TOF) analyzer.
    Each quadrupole has 4 rods, two with positive charge and other two with negative charge. Rods carry DC charges along with alternative current (AC).

    Entry and isolation: Charged peptides enter and feel the charges on rod. By controlling voltage, mass of peptide to be isolated is selected. Most of peptide has unique mass therefore only specific type of peptide with particular mass will pass through the first quadrupole to second. Other peptides collide with walls and rods, and don't enter the second quadrupole.

    Second quadrupole entry: Many of the copies of same peptide enter through small hole in first quadrupole to second quadrupole. Inert gases like nitrogen or argon are introduced to second quadrupole. Peptides collide with the gases molecules and break into fragments. Collisions made are infrequent to avoid breaking down of peptide into individual amino acids. The fragments produced don't have same charge. The two fragments produced from single peptide, the right fragment has positive charge and left has no charge.

    Mass Measure: Then peptide fragments are entered into TOF section which has a plate of strong positive charge. The positive charge fragment gets repelled from plate and fall down, where as no charge fragment pass by the repeller plate.

    Measure of Mass:
    Time taken for the fragment to travel from repeller plate to detector at end is observed. Heavier fragment has more inertia then lighter ones. So the heavier fragments don't react quickly to charge on the plate. Hence lighter fragments move faster then heavier.

    Record Data: The graph with horizontal axis shows TOF. Hence represent mass. Vertical axis represents intensity.

  5. Informatics

    It is the use of computer and database to identify protein. In the graph each peaks represents one peptide fragments i.e. millions of fragments of same mass, hence same sequence. Here 842 represents entire polypeptide with 7 amino acids and 743 represents peptide with 6 amino acid and so on.

    Identify Amino Acid: It is done by simple arithmetic. Here subtract 743 from 842 to get 99 i.e. mass of valine.

    Protein Identification: Sequence is compared with the database to identify the protein. Since entire human genome sequence is known almost every protein can be identified. If no matches are found then a new peptide is discovered.